Hayward Rebecca Frank, Brooks F Phil, Yang Shang, Gao Shiqiang, Cohen Adam E
School of Engineering and Applied Sciences, Harvard University, Cambridge, MA 02138.
Department of Chemistry, Harvard University, Cambridge, MA 02138.
bioRxiv. 2023 Sep 14:2023.02.07.527404. doi: 10.1101/2023.02.07.527404.
Many channelrhodopsins are permeable to protons. We found that in neurons, activation of a high-current channelrhodopsin, CheRiff, led to significant acidification, with faster acidification in the dendrites than in the soma. Experiments with patterned optogenetic stimulation in monolayers of HEK cells established that the acidification was due to proton transport through the opsin, rather than through other voltage-dependent channels. We identified and characterized two opsins which showed large photocurrents, but small proton permeability, PsCatCh2.0 and ChR2-3M. PsCatCh2.0 showed excellent response kinetics and was also spectrally compatible with simultaneous voltage imaging with QuasAr6a. Stimulation-evoked acidification is a possible source of disruptions to cell health in scientific and prospective therapeutic applications of optogenetics. Channelrhodopsins with low proton permeability are a promising strategy for avoiding these problems.
Acidification is an undesirable artifact of optogenetic stimulation. Low proton-permeability opsins minimize this artifact while still allowing robust optogenetic control.
许多通道视紫红质对质子具有通透性。我们发现，在神经元中，高电流通道视紫红质CheRiff的激活会导致显著的酸化，树突中的酸化速度比胞体更快。在HEK细胞单层中进行的模式光遗传学刺激实验表明，酸化是由于质子通过视蛋白运输，而不是通过其他电压依赖性通道。我们鉴定并表征了两种视蛋白，它们表现出大的光电流，但质子通透性小，即PsCatCh2.0和ChR2-3M。PsCatCh2.0表现出优异的响应动力学，并且在光谱上也与使用QuasAr6a进行同步电压成像兼容。在光遗传学的科学和潜在治疗应用中，刺激诱发的酸化可能是细胞健康受到破坏的一个来源。质子通透性低的通道视紫红质是避免这些问题的一种有前景的策略。
酸化是光遗传学刺激中一种不良的假象。低质子通透性视蛋白可将这种假象降至最低，同时仍允许进行强大的光遗传学控制。
